Other epithelial structures which include the liver and pancreas. Numerous non-cystic manifestations for instance cardiac valve abnormalities, diverticular illness, and intracranial aneurysms have been reported (2). Mutations in PKD2 account for 15 of all individuals with ADPKD. The PKD2 protein, polycystin-2 (PC2), is really a Sort II membrane protein of 968 amino acids in length (3). PC2 has the properties of a high-conductance nonselective Ca2 -permeable cation channel. Due to BIO-1211 Autophagy important homology, PC2 (or TRPP2) has been integrated inside the TRP (transient receptor potential) superfamily of channels, which broadly function as cellular sensors for multiple stimuli (4, 5). There’s evidence that PC2 could transduce a mechanosensitive Ca2 present in major cilia (six) although it’s unclear regardless of whether the mechanosensor is PC1, PC2, or yet another protein. Even so, it has also been reported that PC2 can function downstream of G proteincoupled receptor and/or receptor-tyrosine kinase activation at the cell surface (7). The basolateral localization of PC2 in kidney tubules and cells has implicated a possible function in cellcell or cell-matrix adhesion in association with PC1 (ten, 11). Finally, it has been reported that PC2 can function as an endoplasmic reticulum-located Ca2 release channel in some systems (12). Previously we demonstrated that PC2 can exist as PC1-PC2 heterodimers also as PC2 homodimers in native tissues (ten). Interactions involving PC1 and PC2 may well regulate their trafficking and there is evidence for reciprocal activation or inhibition of activity in various experimental systems (13, 14). PC2 might also heterodimerize with TRPC1 via its C terminus (five, 9). PC2-TRPC1 heteromultimers have been shown to possess distinct channel properties from PC1-PC2 heterodimers, being activated in response to G protein-coupled receptor activation within the kidney epithelial cell line, mIMCD3 (9). In yeast twohybrid assays, PC2 can homodimerize via a C-terminal domain, which is distinct from heterodimerization sequences for PC1 or TRPC1 interactions (5, 15). In this report, we describe the identification and functional characterization of a second dimerization domain for PC2 within the N terminus and propose a likely homotetrameric model for PC2 depending on C- and 196309-76-9 References N-terminal interactions. Yeast vectors pGBAD-B and pACT2-B were obtained from D. Markie (University of Otago, NZ) (16). The plasmids LDR and CF applied for the FKBP-FRB dimerization program were gifts of T. Meyer (Stanford University) (17). Generation of PKD2 Plasmids–Unless otherwise stated, the PKD2 plasmids used within this function have been previously reported (18, 19). N-terminal HA-tagged full-length and mutant (L703X) PKD2 constructs had been designed by replacing an XbaI and SacII fragment of a wild-type PKD2 plasmid (gift of S Somlo, Yale University) using the same fragment excised from the previously described HA-L224X plasmid (19). A C-terminal HA-tagged PKD2 mutant construct, R742X, was generated by PCR employing the wild-type PKD2Pk plasmid as a template which includes the HA epitope tag sequence and in-frame cease codon within the reverse primer. The missense PKD2 mutation, D511V, was made by site-directed mutagenesis within the PKD2Pk plasmid template applying a previously published protocol (19). The N-terminal Myc-tagged L224X plasmid was generated by PCR and subcloned into the XbaI and HindIII internet sites of pcDNA3.1 . The plasmids CFP-PKD2-(177) and CFP-PKD2-(123) were generated by fusing the N-terminal sequences of PKD2 in-frame wi.
